As an alternative to solve many of the problems associated with diagnostic use of “spotted arrays” of oligonucleotides (the problems are outlined in “Multianalyte Molecular Analysis Using Application-Specific Random Particle Arrays,” U.S. application Ser. No. 10/204,799, filed on Aug. 23, 2002; WO 01/98765) preferred arrays are formed by binding oligonucleotide probes to encoded microbead particles, including, encoded particles made of polymer resin. See U.S. patent application Ser. No. 10/271,602 “Multiplexed Analysis of Polymorphic Loci by Concurrent Interrogation and Enzyme-Mediated Detection,” filed Oct. 15, 2002, and Ser. No. 10/204,799 supra. The encoded particle-probeconjugates are then assembled in a 2D array format and placed in contact with samples anticipated to contain target polynucleotides with subsequences complementary to the probes, where the target polynucleotides in the samples were previously fluorescently labeled. Binding between the probes and targets is determined by the presence of a fluorescent assay signal. Particular probes generating a positive assay signal can be determined by decoding the array.
There are several known and commercially available methods for attachment of oligonucleotide probes to microbeads. A great number of covalent immobilization schemes for oligonucleotide probes to microparticles have been devised and are available either in open literature or commercially. Traditional covalent immobilization techniques use functionalized beads (i.e, beads functionalized with reactive groups like amino, carboxyl, tosyl, aldehyde, epoxy, hydrazide and others) to link to complementary functional groups on the end of oligonucleotide probes (Maire K. Walsh, Xinwen Wang and Bart C. Weimer, Optimizing the immobilization of single-stranded DNA onto glass beads, J. Biochem. Biophys. Methods 2001; 47:221-231). Often times such binding protocols lead to improper orientation and steric hindrance problems. The hybridization performance of such covalently immobilized probes can be improved by introduction of spacer molecules (Edwin Southern, Kalim Mir and Mikhail Shchepinov; Molecular Interactions on Microarrays. Nature Genetics Supplement, 21, 1999, pp. 5-9), however, implementation is often difficult and impractical.
A practical and robust probe binding chemistry is therefore important for the optimal performance of a microbead array based assay. The chemistry must allow the probes to bind to the particles with high efficiency, in order to maintain a consistent concentration of probes on the bead surface and also the reaction must not alter the efficiency of probe-target binding. Moreover, the reaction must have minimum batch to batch variability. In one commonly used method, functionalized microparticles are coated with Neutravidin (Pierce, Rockford, Ill.), streptavidin or avidin, which are biotin binding proteins, to mediate immobilization of biotinylated probes. The avidin-biotin interaction is highly specific and one of the strongest known (with an association constant (KA) of the order of 1015M−1 in aqueous solutions) and provides nearly irreversible linkage between the bead surface immobilized protein and the biotinylated probe molecule. See U.S. patent application Ser. No. 10/271,602, supra. The method described below for binding probes to polyelectrolytes are preferred to these known methods, because they were demonstrated as capable of inducing attachment of greater numbers of oligonucleotides to beads.